Imbalances in cortical dopamine receptor (DR) mediated signaling are believed to underly both the cognitive and psychotic aspects of schizophrenia. Using yeast two-hybrid screens, we identified several DR-interacting proteins (DRIPs) that are prominently involved in intracellular calcium (Ca++) homeostasis (calcyon, NCS-1, TRPC-1, and CAPS). Disease-related increases in calcyon and NCS-1 protein levels were detected in dorsolateral prefrontal cortex (DLPFC) of patients with schizophrenia. We propose to extend our discovery based research on DR signaling complexes (DRSCs) guided by the hypothesis that abnormal dopamine-mediated Ca++ signaling is the basis of prefrontal dysfunction in schizophrenia. The possibility that this approach may also identify potential schizophrenia susceptibility genes (SSGs) is supported by a recent genetic association study which found evidence for an SSG at 10q26, the chromosomal position of the calcyon gene. We will utilize MALDI TOF-TOF mass spectrometry to identify the regional and receptor-specific composition of D1 and D2 DR-containing DRSCs immunoprecipitated from rat and monkey prefrontal cortex and striatum. It is likely that among the DRSC components, some proteins will interact directly with the receptor (DRIPs), while others (DRAPs or DR associated proteins) may interact with peripheral components of the complex. We will confirm interaction of DRSC proteins using a combination of biochemical and immunohistochemical methods. The effects of DRIP and DRAP interactions on DR function will be studied in transfected mammalian cells and native neural systems with a focus on Ca++ signaling and homeostasis. Expression of DRIPs and DRAPs will be analyzed in collections of schizophrenic brains. We will generate transgenic mice overexpressing NCS-1 or calcyon within the prefrontal cortex to learn whether these candidate schizophrenia-associated DRIPs are involved in the etiology of the disease. Transgenic mice will be subjected to a battery of behavioral, anatomical, and Physiological tests relevant to prefrontal deficits manifested in schizophrenia in collaboration withother subprojects. Identifying previously unknown proteins directly or indirectly associated with DRs should provide new insights into mechanisms of dopaminergic signaling in DLPFC, and clues as to the etiology of natomical and physiological defects manifested in schizophrenia.